Sensor

ABSTRACT

There is disclosed a sensor for an analyte comprising a working electrode assembly, itself comprising a micro-electrode array in which each micro-electrode is coated with a layer of redox state dependent conducting organic polymer, a counter electrode; a conductive medium containment into which the electrodes are disposed and a material for analysis may be introduced; means for applying an alternating electric polarizing potential across the electrodes; and means for detecting a variation in a conductimetric property across the {working electrode→counter electrode} plane in the presence of the analyte.

This invention relates to sensors for analytes present in solution.

Conductimetric sensors based on redox state dependent conducting organicpolymers such as polypyrrole, polyaniline and polyindole are well known.Such sensors commonly comprise a layer of the conducting organic polymerbridging two closely spaced electrodes, and commonly rely upon thedetection of changes in the resistance or ac impedance of the polymer.This change in resistance is caused by the interaction between theanalyte--which may be present in solution or in the gas phase--and thepolymer. An important area of application lies in the incorporation ofenzymes into the organic polymer to produce `biosensors` capable ofquantitative sensing of biologically significant analytes. Exposure ofthe enzyme to the analyte induces catalytic activity which in turninduces conductivity changes within the enzyme trapping polymer matrix.Although the precise nature of the conductivity changes are unclear--andmay well differ with different combinations of polymer and enzyme--localH⁺ concentration effects and, in the case of oxidases, ketones andaldehydes, H² O² generation have been implicated. Broadly speaking, theconductivity of the polymer is perturbed by the redox behaviour of theenzyme on exposure to the analyte. Since enzyme-substrate interactionsare typically very specific, the associated biosensor represents ahighly selective method of analyte detection.

However, it is usually only possible to bridge the electrodes byelectrochemical growth of organic polymer if the electrodes are ratherclosely spaced (typically <20 μm). This upper limit imposes restrictionson the magnitude of the measured signal and also requires preciseelectrode geometries which in turn requires technically demanding andexpensive fabrication procedures such as screen printing or silicon chipfabrication techniques.

An appealing alternative which circumvents the problem of preciseelectrode alignment would involve measuring the impedance or admittanceof a polymer bridging an analyte solution and a planar workingelectrode; in such an arrangement measurements would be made in the{working electrode→polymer→analyte solution→counter electrode} plane.Detection of the analyte would be accomplished by detecting changes inthe impedance or admittance of the polymer induced by interactionbetween the polymer and the analyte. However, the linear mass transportregime operating in this arrangement would inevitably result in adetection signal of insufficient magnitude for useful measurement.

The present invention is based upon the concept of incorporating withinthe above described arrangement a working electrode assembly whichcomprises an array of conducting organic polymer coatedmicro-electrodes. Such an electrode results in radial transport of theanalyte, rendering feasible the use of the arrangement as a sensor.

The present invention further provides examples of such arrays ofmicro-electrodes and methods of fabricating same.

International Publication WO 91/08474 describes a method for fabricatinga microelectrode by photoablation. The microelectrode was used in anamperometric assay method for the detection of heavy metals.

According to one aspect of the invention there is provided a sensor foran analyte comprising a working electrode assembly itself comprising amicro-electrode array in which each micro-electrode is coated with alayer of redox state dependent conducting organic polymer; a counterelectrode; a conductive medium containment into which the electrodes aredisposed and a material for analysis may be introduced; means forapplying an alternating electric polarising potential across theelectrodes; and means for detecting a variation in a conductimetricproperty across the {working electrode→counter electrode} plane in thepresence of the analyte.

The conductive medium containment may comprise a conducting solution.

The conductimetric property may be the impedance across the {workingelectrode→counter electrode} plane.

The variation in the conductimetric property may be detected as afunction it of the applied frequency.

Alternatively, said variation may be detected at a single appliedfrequency.

The working electrode may be fabricated by coating a planar electrodewith an insulating polymer, sonically ablating the insulating polymercoating to produce a plurality of micro-pores, and depositing conductingorganic polymer into said micro-pores. The conducting organic polymermay be deposited electrochemically or by other means such as chemicalvapour deposition, photopolymerisation techniques or other thin or thickfilm surface coating technologies.

An enzyme may be entrapped within the semiconducting organic polymermatrices. The semiconducting organic polymer may be polyaniline,polypyrrole. polyindole or equivalents thereof. The enzyme may be aredox enzyme such as glucose oxidase, alcohol oxidase and alcoholdehydrogenase. Further, the planar electrode may be a noble metal or aconducting material such as carbon and the insulating polymer may bepolydiaminobenzenedihydrochloride, Teflon, PVC or polyethylvinylbenzene.

According to a second aspect of the invention there is provided a methodof fabricating a working electrode assembly comprising coating a planarelectrode with an insulating polymer, sonically ablating the insulatingpolymer coating to produce a plurality of micro-pores, and depositingredox state dependent conducting organic polymer into said microphores.

The organic polymer may be deposited electrochemically, by chemicalvapour deposition, photopolymerisation techniques or other thin or thickfilm surface in coating technologies.

An enzyme may be entrapped within the organic polymer matrices.

Sensors and methods of fabricating working electrode assemblies inaccordance with the invention will now be described with reference tothe accompanying drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

    ______________________________________                                        Figure 1 is a schematic illustration of the electrode arrangement:            Figure 2 is a schematic illustration of the control arrangement:              Figure 3 shows a possible equivalent circuit; and                             Figure 4 is a graph of typical log (impedance) vs log (frequency)                      profiles at different analyte concentrations.                        ______________________________________                                    

PREFERRED EMBODIMENTS

FIGS. 1 and 2 depict a sensor for detecting an analyte according to thepresent invention, which comprises a working electrode assembly 10, acounter electrode 12 and a conducting solution 14, wherein the workingelectrode assembly 10 comprises a micro-electrode array in which eachmicro-electrode is coated with a layer of redox state dependentconducting organic polymer. The sensor further comprises a frequencyresponse analyser 16 which i) applies an alternating electric polarisingpotential across the electrode arrangement 18 and ii) detects anyvariation in a chosen conductimetric property across the {workingelectrode substrate→polymer→solution→counter electrode} plane when theanalyte is present in the solution. The variation may alternatively bedetected by a phase sensitive detector. Control of the frequencyresponse analyser and collection of data may be effected inter alia by acomputer 20 via a suitable interface.

The conductimetric sensor of the present invention utilises redox statedependent conducting organic polymer as the active, analyte sensingmedium, but represents a very different detection arrangement to priorart conductimetric devices which employ polymers of this type. Inparticular, prior art devices typically comprise closely spacedelectrodes bridged by conducting organic polymer, close in this contextbeing about 15-20 μm or less. This approximate upper limit is due todifficulty of effectively bridging greater electrode separations withelectrochemically grown polymer--by far the most commonly employedmethod of depositing the conducting polymer. Physically, the reason forthis difficulty is that the separation of the electrodes exceeds thelength of the polymeric chains, resulting in very high resistances andpoor mechanical qualities. Consequently, precise electrode geometriesare required necessitating the use of technically demanding andexpensive fabrication procedures such as screen printing or silicon chipfabrication techniques.

The arrangement of the present invention does not utilise conductingpolymer as an electrode bridge, and thus does not require precisealignment of the electrodes. Furthermore, it is noted that use of aconventional planar working electrode coated with semiconducting organicpolymer would result in linear mass transport of the analyte, which inturn would result in an unfeasibly small detection signal. With theintroduction of micro-electrodes the mass transport of analyte becomesradial in nature, permitting analyte detection with excellentsensitivity.

Detection of analyte may be achieved by detecting changes in the complexplane impedance between the counter electrode and the array of polymercoated micro-electrodes which comprise the working electrode. However,detection of other conductimetric properties, such as conductance orsusceptance, are also within the scope of the invention. In any event,it is necessary to separate the desired measurement of analyte inducedchanges in, for example, polymer resistance from the otherconductimetric components of the system. FIG. 3 shows a Randlesequivalent circuit, which indicates that the conductimetric behaviourbetween the counter electrode and the working electrode may bedecomposed into:

    ______________________________________                                        i)     the uncompensated solution resistance R.sub.u,                         ii)    the capacitance due to the electrical double layer C.sub.dl,           iii)   the reaction resistance, which may in turn comprise a                         Warburg impedance W and a charge transfer resistance R.sub.ct,         iv)    the capacitance of the polymer layer C.sub.pf.                         ______________________________________                                    

The Warburg impedance W is negligible at high excitation frequencies(typically >100 Hz) and thus R^(ct) and C^(pf), which are modulated bythe analyte-polymer interaction, may be extracted.

A schematic illustration of the way in which impedance may vary as afunction of applied frequency and analyte concentration is shown in FIG.4. The response curves a, b, c, d are in order of increasing analyteconcentration. The presence of analyte may be detected by monitoringchanges in the response function over a range of applied frequencies.Alternatively, it may prove expedient to detect changes in impedance ata fixed frequency, this frequency advantageously being one at whichparticularly large impedance changes are induced by the analyte-polymercombination in question, such as the frequency marked A in FIG. 4. Thecomputer 20 may be used to store the response or response functionobtained with no analyte present in the solution 14 as a referencesignal, and to compare this reference signal with the response orresponse function obtained in the presence of an analyte.

Fourier transform analysis techniques are also applicable. However, itshould be noted that a dc measurement of bulk sensor resistance wouldnot suffice, since the dominant contribution to this resistance would bedue to charging of the capacitances within the circuit, giving rise to adrifting value.

The invention also provides examples of arrays of semiconducting organicpolymer coated micro-electrodes and methods of fabricating same.Generally speaking, working electrodes of the present invention may befabricated by coating a planar electrode with an insulating polymer,sonically ablating the insulating polymer coating to produce a pluralityof micro-pores, and depositing conducting organic polymer into themicro-pores. such that an electrically parallel connectedmicro-electrode array is formed. It has been shown (N A Madigan, C R SHagan and L A Coury, J. Electrochem. Soc., 141 (1994) 1014) thatmicro-electrode arrays may be produced by sonicating insulating polymercoated electrodes, but the coating of the micro-electrodes withconducting organic polymer and the use of such a micro-electrode arrayas a sensor has no antecedent. Examples of insulating polymer filmsinclude polydiaminobenzenedihydrochloride, PVC, Teflon,polyethylvinylbenzene and the like. Examples of substrate electrodematerials include gold, platinum, ruthenium, gold/platinum alloys andglassy carbon. Commonly employed solvents such as water, decane anddioxane may be used for sonication.

The conducting organic polymer may be deposited electrochemically, theprecise details of the polymerisation process not varying substantiallyfrom well known literature methods. (see for example J C Cooper and E AH Hall. Biosensors & Bioelectronics, 7 (1992) 473) Examples ofelectropolymerisable conducting organic polymers include polyaniline,polypyrrole, polyindole and poly-N-methylpyrrole, although it is to beunderstood that this list is a non-limiting one: many examples ofsuitable conducting organic polymers may be found in the literature.Other deposition means, such as chemical vapour deposition,photopolymerisation techniques or other thin or thick film surfacecoating technologies are within the scope of the invention.

An important aspect of the present invention lies in the incorporationof enzymes into the conducting organic polymer to produce biosensorscapable of sensing of, for example, biologically significant analytes.The incorporation of enzymes into the polymer may be accomplished by anyof the many well characterised methods documented in the literature. Forexample, the polymer may be electrochemically polymerised from asolution containing both monomer and enzyme.

A specific example of a sensor of the present invention is a devicesensitive to the presence of glucose. The working electrode was producedby sonication in water of a gold planar electrode coated withpolydiaminobenzenedihydrochloride, followed by electropolymerisation ofaniline in the presence of glucose oxidase. Sonication is typically at15 KHz for one minute. Glucose oxidase catalyses the oxidation ofglucose, i.e: ##EQU1##

The choice of polyaniline as the conducting organic polymer isinfluenced by its stability in the presence of hydrogen peroxide.However, it should be noted that the use of other conducting organicpolymers and other enzymes is within the scope of the invention.Measurements of impedance profile as a function of applied frequency atvarying concentrations of the glucose analyte (1-20 mM glucose in a pH 7phosphate buffer) have been made using a glucose sensor of this type.The results indicate that change in an impedance value measured at achosen frequency (compared to the impedance obtained at the samefrequency with no glucose present) is proportional to the concentrationof glucose in the solution in the low (<5 mM) concentration regime. Asan example, measurements made at a frequency of 11.3 Hz show that theimpedance of the polymer film is ca. 400 Ω in the absence of glucose,but in the presence of a 5 mM glucose solution this impedance drops toca. 200 Ω. This represents an extremely large signal, well above thedetection limit of the apparatus employed, which is capable of detectingchanges in resistance of the order of 0.1 Ω or less.

Similar results have been obtained with a sensor incorporating alcoholoxidase as the enzyme; ethanol is the analyte in this instance. Thisdemonstrates that the present invention may be applied to differentanalyte and enzyme combinations. For ethanol concentrations of 15 mM, adrop in impedance from ca. 400 Ω to ca. 200 Ω has been observed.

Many further variations within the scope and spirit of the invention arepossible. For instance, it may be desirable to combine several workingelectrodes within one device, wherein each working electrode comprises adifferent polymer and/or enzyme. In one example each working electrodewould have a different enzyme incorporated into the polymer matrix, andeach enzyme would be sensitive to a different analye. The resultingdevice would be sensitive to the presence of a number of specificanalytes. Alternatively, it is well known that semiconducting organicpolymer based conductimetric sensors which do not incorporate analytereception agents such as enzymes are usually sensitive to a range ofanalytes. Therefore, with prior art devices which rely upon themeasurement of changes in dc resistance, precise identification of ananalyte with a single sensor incorporating a single polymer can presenta problem. The present invention is advantageous in this respect, sincethe response of the sensor as a function of frequency can provide anextra detection dimensionality compared to a single, bulk, dc resistancemeasurement, and may be considered a `fingerprint` of the analytedetected. However, it may be desirable to produce a device having aplurality of working electrodes which each involve a differentsemiconducting organic polymer; in such a device the pattern of responseacross the plurality of working electrodes may be indicative of aparticular analyte. Indeed, it may prove possible to deduce theindividual components of a mixture from the response patterns of such adevice.

What is claimed is:
 1. A sensor for an analyte comprising:a workingelectrode comprising a conductive electrode having an insulating layerthereon, said insulating layer having a plurality of microporestherethrough, and a conducting organic polymer deposited in saidmicropores, said polymer in said micropores being electricallyinterconnected by said conductive electrode to form a microelectrodearray; a counter electrode; a conductive medium containment into whichthe electrodes are disposed and a material for analysis may beintroduced; means for applying an alternating electric polarizingpotential across the electrodes; and means for detecting a variation ina conductimetric property across a working electrode-counterelectrodeplane in the presence of the analyte.
 2. A sensor according to claim 1in which the conductive medium containment comprises a conductingsolution.
 3. A sensor according to claim 1 or claim 2 in which theconductimetric property detected is the impedance across the {workingelectrode→counter electrode} plane.
 4. A sensor according claim 1 inwhich the variation in the conductimetric property is detected as afunction of the applied frequency.
 5. A sensor according claim 1 inwhich the variation in the conductimetric property is detected at asingle applied frequency.
 6. A sensor according claim 1 in which theworking electrode is fabricated by coating a planar electrode with aninsulating polymer, sonically ablating the insulating polymer coating toproduce a plurality of micro-pores, and depositing redox state dependentorganic polymer into said micro-pores.
 7. A sensor according to claim 6in which the conducting organic polymer is deposited electrochemically,by chemical vapour deposition or by photopolymerization.
 8. A sensoraccording claim 6 in which an enzyme is entrapped within the conductingorganic polymer matrices.
 9. A sensor according to claim 8 in which theenzyme is glucose oxidase, alcohol oxidase or alcohol dehydrogenase. 10.A sensor according to any of claims 6 to 9 in which the insulatingpolymer is polydiaminobenzenedihydrochloride, Teflon, PVC orpolyethylvinylbenzene.
 11. A method of fabricating a working electrodeassembly comprising coating a planar electrode with an insulatingpolymer, sonically ablating the insulating polymer coating to produce aplurality of micro-pores, and depositing redox state dependentconducting organic polymer into said micro-pores.
 12. A method accordingto claim 11 in which the organic polymer is deposited electrochemically,by chemical vapour deposition or by photopolymerization.
 13. A methodaccording to claim 11 or claim 12 in which an enzyme is entrapped withinthe organic polymer matrices.